Scanning spectrophotometer for high throughput fluorescence detection and fluorescence polarization

ABSTRACT

A fluorescence spectrophotometer system may be implemented in scanning fluorescence polarization detection applications. A wavelength and area scanning fluorescence spectrophotometer system may include a light source, an excitation double monochromator, an excitation/emission light transfer module, an emission double monochromator, a high speed timer-counter circuit board, a precision positioning apparatus for positioning a sample relative to the focal plane of the excitation light, and polarizing filters at the excitation side and the emission side. The system may be operative to analyze more than one fluorescent compound in the sample; additionally or alternatively, the system enables analysis of samples from selected ones of a plurality of samples.

The present application claims the benefit of a provisional application,Ser. No. 60/259,326, filed Dec. 29, 2000, entitled “SCANNINGSPECTROPHOTOMETER FOR HIGH THROUGHPUT FLUORESCENCE DETECTION ANDFLUORESCENCE POLARIZATION.”

TECHNICAL FIELD

Aspects of the present invention relate generally to wavelength scanningfluorescence spectrophotometers using dual grating monochromators, butnot optical filters, to select excitation and emission wavelengths oflight and to detect and to quantify simultaneous fluorescence emmission,including polarized fluorescence emission, from multiple fluorophores inthe same sample.

BACKGROUND Definitions

1) FLUORESCENCE: The result of multi-stage process of energy absorptionand release by electrons of certain naturally occurring minerals,polyaromatic hydrocarbons and other heterocycles.

2) EXCITATION: Photons of energy, e=hv_(exc), are supplied by a lightsource and absorbed by an outer electron of a fluorophore, which iselevated from the ground state, S₀, to an excited electronic singletstate, S′₁.

3) EXCITED STATE LIFETIME: An excited electron remains in the singletstate for a finite period, typically from 1 to 20 nanoseconds, duringwhich the fluorophore undergoes a variety of changes includingconformational changes and alterations in the interaction with solvent.As result of these changes, the energy of the S₁, singlet electronpartially dissipates to a relaxed singlet excited state, S₁ from whichfluorescence emission of energy occurs, returning the electron to theground state, S₀.

4) EMISSION: Photons of energy, e=h_(em), are released from an excitedstate electron, which returns the fluorophore to the ground state. Owingto energy loss during the excited state lifetime, the energy of thesephotons is lower than that of the exciting photons, and the emittedlight is of longer wavelength. The difference in the energy (orwavelengths) is called the Stoke's shift and is an important feature inthe selection of a dye for use as a label or in a probe. The greater theStoke's shift, the more readily low numbers of photons can bedistinguished from background excitation light.

5) FLUOROPHORES: Fluorescent molecules are generally referred to asfluorophores. When a fluorophore is utilized to add color to some othermolecule, the fluorophore is called a fluorescent dye and thecombination is referred to as a fluorescent probe. Fluorescent probesare designed to: 1) localize and help visualize targets within aspecific region of a biological specimen, or, 2) respond to a specificstimulus.

6) ELECTROMAGNETIC SPECTRUM: The entire spectrum, considered as acontinuum, of all kinds of electric and magnetic radiation, from gammarays, having a wavelength of 0.001 Angstroms to long waves having awavelength of more than 1,000,000 kilometers and including theultraviolet, visible and infrared spectra.

7) FLUORESCENCE SPECTRUM: Unless a fluorophore is unstable(photobleaches), excitation and emission is a repetitive process duringthe time that the sample is illuminated. For polyatomic molecules insolution, discrete electronic transitions are replaced by broad energybands called the fluorescence excitation and fluorescence emissionspectra, respectively.

8) MONOCHROMATOR: A device which admits a wide spectral range ofwavelengths from the electromagnetic spectrum via an entrance aperture,and, by dispersing wavelengths in space, makes available at an exitaperture only a narrow spectral band of prescribed wavelength(s).Optical filters differ from monochromators in that they providewavelength selection through transmittance of selected wavelengthsrather than through spatial dispersion. A second distinguishing featureof a monochromator is that the output wavelength(s), and in many cases,the output spectral bandwidth, may be continuously selectable.Typically, the minimal optical components of a monochromator comprise:

(a) an entrance slit that provides a narrow optical image;

(b) a collimator which ensures that the rays admitted by the slit areparallel;

(c) some component for dispersing the admitted light into spatiallyseparate wavelengths;

(d) a focusing element to re-establish an image of the slit fromselected wavelengths; and,

(e) an exit slit to isolate the desired wavelengths of light.

In a monochromator, wavelength selection is achieved through a drivesystem that systematically pivots the dispersing element about an axisthrough its center. Slits are narrow apertures in a monochromator whichmay have adjustable dimensions. Slits effect selection of the desiredwavelength(s) and their dimensions may be adjustable.

9) DUAL GRATING MONOCHROMATOR: A monochromator containing two gratingscoupled in series. The second grating accepts wavelengths of lightselected by the first and further separates the prescribed wavelengthsfrom undesired wavelengths.

10) WAVELENGTH SCANNING: Continuous change of the prescribed outputwavelength(s) leaving the exit slit of a monochromator. In aspectrophotometer, wavelengths of the electromagnetic spectrum arescanned by the excitation monochromator to identify or prescribe thewavelength(s) at which a fluorophore is excited; wavelength scanning bythe emission monochromator is used to identify and detect thewavelength(s) at which a fluorophore emits fluorescent light. Inautomated fluorescence spectrophotometers, wavelength scanning by theexcitation and emission monochromators may be performed eitherseparately or concurrently (synchronous scanning).

11) AREA SCANNING: Area scanning is distinct from wavelength scanningand is the collective measurement of local fluorescence intensities in adefined two dimensional space. The result is an image, database or tableof intensities that maps fluorescence intensities at actual locations ina two dimensional sample. At its simplest, area scanning may be aphotograph made with a camera in which all data are collectedconcurrently. Alternatively, the sample may be moved past a detectorwhich measures the fluorescence in defined sub-areas of a sample. Thecollected information creates a matrix which relates fluorescenceintensity with position from which an image, table or graphicalrepresentation of the fluorescence in the original sample can becreated.

12) FLUORESCENCE DETECTORS

Five elements of fluorescence detection have been established throughlaboratory use of fluorophores during the last two decades:

(a) an excitation source,

(b) a fluorophore,

(c) some type of wavelength discrimination to isolate emission photonsfrom excitation photons,

(d) some type of photosensitive response element that converts emissionphotons into a recordable form, typically an electronic signal or aphotographic image, and,

(e) a light tight enclosure to restrict ambient light.

Fluorescence detectors are primarily of four types, each providingdistinctly different information:

(a) Cameras resolve fluorescence as spatial coordinates in twodimensions by capturing an image: [a] as a photographic image on highlysensitive film, or, [b] as a reconstructed image captured on arrays ofpixels in a charge coupled device (CCD).

(b) Fluorescence microscopes also resolve fluorescence as spatialcoordinates in two or three dimensions. Microscopes collect all of theinformation for an image for a prescribed visual field at the same timewithout any movement of either the sample or the viewing objective. Amicroscope may introduce qualitative estimation of fluorophoreconcentration through use of a camera to capture an image in which casethe measure is a function of exposure time.

(c) Flow cytometers measure fluorescence per biological cell in aflowing liquid, allowing subpopulations within a mixture of cells to beidentified, quantitated, and in some cases separated. Flow cytometerscannot be used to create an image of a defined area or performwavelength scanning. The excitation light source is invariably a laserand wavelength discrimination is accomplished through some combinationof tunable dye lasers and filters. Although these instruments may employphotomultiplier tubes (PMTs) to detect a measurable signal there are noflow cytometers that employ monochromators for wavelength scanning.

(d) Spectrofluorometers (spectrophotometer(s)) typically employ a PMT todetect fluorescence but can measure either: [a] the average currentevoked by fluorescence over time (signal averaging), or, [b] the numberof photons per unit time emitted by a sample (photon counting).

Fluorescence spectrophotometers are analytical instruments in which afluorescent dye or probe can be excited by light at specificwavelengths, and, concurrently, have its emitted light detected andanalyzed to identify, measure and quantitate the concentration of theprobe. For example, a piece of DNA may be chemically attached, orlabeled, with fluorescent dye molecules that, when exposed to light ofprescribed wavelengths, absorb energy through electron transitions froma ground state to an excited state. As indicated above, the excitedmolecules release excess energy via various pathways, includingfluorescence emission. The emitted light may be gathered and analyzed.Alternatively, a molecule of interest may be conjugated to an enzymewhich can convert a specific substrate molecule from a non-fluorescentto a fluorescent product following which the product can be excited anddetected as described above.

The ranges of excitation and emission wavelengths employed in afluorescence spectrophotometer typically are limited to the ultravioletand visible portions of the electromagnetic spectrum. For the purposesof fluorescence detection, useful dyes are those which are excited by,and emit fluorescence at, a few, narrow bands of wavelengths within thenear ultraviolet and visible portions of the electromagnetic spectrum.Desired wavelengths for excitation of a specific fluorescent moleculemay be generated from

1) a wide band light source by passing the light through a series ofbandpass filters (materials which transmit desired wavelengths of lightand are opaque to others), or cut-on filters (materials which transmitall wavelengths longer or shorter than a prescribed value),

2) a narrow band light source such as a laser, or,

3) an appropriate monochromator.

For a wide band light source, the light to which a fluorescent dye isexposed is typically isolated through bandpass filters to select adesired wavelength from the ultraviolet or visible spectrum for use inexcitation. In monochromator-based instruments, the wavelength of choiceis obtained after light from the source has been dispersed into aspectrum from which the desired wavelength is selected. Whatever thelight source, the fluorescence emission is typically isolated throughbandpass filters, cut-on filters, or emission monochromators to select adesired wavelength for detection by removal of all light of anywavelengths except the prescribed wavelengths. Most fluorescencedetection involves examination of specimens that are in a liquid phase.The liquid can be contained in a glass, plastic or quartz containerwhich can take the form of, for example: an individual cuvette; aflow-through cell or tube; a microscope slide; a cylindrical orrectangular well in a multiwell plate; or silicon microarrays which may,have many nucleic acids or proteins attached to their surfaces.Alternatively, the liquid can be trapped in a two-dimensionalpolyacrylamide or agarose gel. In each of these cases, light which hasalready passed through the optical filters to select the correctwavelengths for excitation illuminates the sample in the container orgel; concurrently, emitted light is also collected, passed through asecond set of optical filters to isolate the wavelengths of emission,and then detected using a camera, or photosensor.

The optical filters used in fluorescence detectors presentcharacteristics that limit the sensitivity, dynamic range andflexibility of fluorescence detection, including: light absorption whichcauses a loss of efficiency through the system; inherentauto-fluorescence, which produces a high background signal; transmissionof other wavelengths outside the wavelengths of desired bandpass which,in turn limits both sensitivity and dynamic range. Optical filters mustbe designed and manufactured to select for discrete ranges ofwavelengths (“center-width bandpasses”) which limits fluorescencedetection to the use of compounds which are excited and emit atwavelengths appropriate for those filters. Development of a newfluorescent dye with unusual spectral properties may necessitate designof a new excitation/emission filter pair.

To increase efficiency in fluorescence cuvette spectrophotometers aswell as to provide continuous selection of wavelengths, it has beenknown to use grating or prism-based monochromators to disperse incominglight from an excitation source, select a narrow band of excitationwavelengths and, separately, to select an emission wavelength. Gratingscome in many forms but are etched with lines that disperse broadbandlight into its many wavelengths. A monochromator typically includes alight-tight housing with an entrance slit and an exit slit. Light from asource is focused onto the entrance slit. A collimating mirror withinthe housing directs the received beam onto a flat optical gratin, whichdisperses the wavelengths of the light onto a second collimating mirrorwhich in turn focuses the now linearly dispersed light onto the exitslit. Light of the desired wavelength is selected by pivoting thegrating to move the linear array of wavelengths past the exit slit,allowing only a relatively narrow band of wavelengths to emerge from themonochromator. The actual range of wavelengths in the selected light isdetermined by the dimensions of the slit. The process of continuousselection of a narrow band of wavelengths from all wavelengths of acontinuous spectrum is referred to as wavelength scanning and the angleof rotation of the dispersing optical grating with respect to theentrance and exit slits correlates with the output wavelength of themonochromator. In order to select the wavelengths of excitation andfluorescence detection, it has been known to use two gratings in eachmonochromator to enhance wavelength selection for both the excitationand emission light in a fluorescence spectrophotometer. While themonochromators potentially eliminate the need to use optical filters forwavelength selection and free the scientist from the limitations offilters, their use imposes other limitations on instrument sensitivityand design. For example, monochromators having the configurationsdescribed above have the disadvantage of requiring at least four mirrorsand two dispersing elements, along with associated light blockingentrance and exit slits. Consequently, such devices have been relativelycomplex and comparatively inefficient compared to filter basedinstruments.

Analysis of multiple samples in multi-well plates is a highlyspecialized use of fluorescence spectrophotometers. Typically, theexcitation light is introduced into a well from a slight angle above thewell in order to allow the majority of the fluorescence emission lightfrom the sample within a well to be collected by a lens or mirror.However, as the number of wells per plate is increased (e.g., from 96wells per plate to in excess of 9600 per plate), this side illuminationconfiguration becomes disadvantageous, since most of the incomingexcitation light strikes the side of the well rather than the sample.Since such wells typically have black side walls, much of the excitationlight is lost.

As mentioned above, one method employed to overcome the limitations ofside illumination configurations has been use of an optical fiber toguide the excitation light to an illumination end of the fiber directlypositioned over a well. A second bundle of fibers is employed to collectlight from the well and transmit it to the PMT. In a variation of thisdesign, a bifurcated optical fiber positioned above a microwell has beenused to carry light both into and out of the well. However, opticalfibers typically introduce absorption losses and may also auto-fluoresceat certain wavelengths. Accordingly, such a solution is not particularlyefficient.

Another approach has been to use multi-well plates with transparentbottoms, and exposing a sample within a well to excitation light fromthe bottom while collecting emission light from the open top. While thisapproach has value in some circumstances, light is lost from absorptionas well as from light scattering by the plastic at the well bottom.Additionally, the transparent plate material may itself auto-fluoresce.In addition, well-to-well optical reproducibility of the well bottommaterial has not been achieved, which has limited the ability tocorrelate measurements on a well-to-well or plate-to-plate basis.Accordingly, such a solution has proven to be less efficient thanilluminating and collecting light from the same side of a sample.

Examples of such prior art using fiber optic light paths include asingle unit fluorescence microtiter plate detector (the “SpectromaxGEMINI”) introduced in 1998 by Molecular Devices, which employs a hybridcombination of single grating monochromators, filters, mirrors andoptical fibers, and the “Fluorolog-3”, a modular instrument, and the“Skin-Sensor”, a unitized instrument, both produced by Instruments SA,both of which employ bifurcated fiber optic bundles to conduct lightfrom an excitation monochromator and to collect light from a sampleafter which it is transmitted to the excitation monochromator.

It should be noted that microtiter plate applications of fluorescencemonochromators are also limited to microwell plates with 384, 96, orfewer wells; that is, 1536-well microplates, as well as “nanoplates”containing 2500 wells, 3500 wells, and even 9600 wells cannot be usedwith the current fiber-optic/monochromator based instruments. Detectorsfor such plates typically use lasers and filters combined with confocalmicroscopy.

For the large number of applications involving glass microscope slides,polyacrylamide gels or standard 96-well, 384-well and 1536-wellmicrowell plates, it would be desirable to have a fluorescencespectrophotometer that provides high efficiency, enables high precisioncontinuous excitation and emission wavelength selection, providessignificantly greater dynamic range, eliminates the use of opticalfilters and optical fibers (i.e., light paths do not pass through anyoptical materials other than air), and has a highly efficient structurefor both guiding the excitation light onto a sample and collecting theemission light from the sample in a microtiter well or on a twodimensional surface such as a glass microscope slide, polyacrylamide gelsilicon microarray, or other solid surfaces.

In general the measurement of fluorescent light intensity, theluminescence, is defined as the number of photons emitted per unit time.Fluorescence emission from atoms or molecules can be used to quantitatethe amount of an emitting substance in a sample. The relationshipbetween fluorescence intensity and analyte concentration is:

F=kW _(e) P _(o)(1−10^((εbc)))

where F is the measured fluorescence intensity, k is a geometricinstrumental factor, Q_(e) is the quantum efficiency (photonsemitted/photons absorbed), P_(o) is the probability of excitation whichis a function of the radiant power of the excitation source, ε is thewavelength dependent molar absorptivity coefficient, b is the pathlength, and c is the analyte concentration. In previous applications,the above equation was simplified by expanding the equation in a seriesand dropping the higher terms to give:

F=kQ _(e) P _(o)(2.303*ε*b*c).

In the past, this relationship was acceptable because fluorescenceintensity appeared to be linearly proportional to analyte concentration.The equation fails, however, to provide for true comparison of thefluorescence intensities of different fluorophores because measurementof fluorescence intensity is highly dependent upon k, the geometricinstrumental factor.

Different types of detectors vary in both the time period during which ameasurement is made and the speed at which each can discriminate betweenphotons, characteristics which can be of critical importance whencomparing the luminosity of two fluorophores. Consider two fluorescentdyes that differ only in that the excited state lifetime of one istenfold longer than that of the excited state lifetime of the secondfluorophore (e.g. 1 nanosecond and 10 nanoseconds, respectively). Whendetected using photographic film exposed for a defined exposure time,the dye with the shorter lifetime would clearly appear brighter on theexposed film. However, if a detector employing continuous excitationwere used which could not discriminate between photons at a high enoughfrequency, the fluorophore with the shorter excited state lifetime couldactually be emitting far more photons but the detector could erroneouslyindicate that the fluorescence intensity of the two dyes was the same.

Fluorescence is detected in spectrophotometers through generation ofphotocurrent in an appropriate photosensitive device such as a PMT orother photosensing device, both of which are characterized by low levelsof background or random electronic noise. For this reason, fluorescentemission processes are best characterized by Poisson statistics andfluorescence can be measured through either photon counting or signalaveraging:

Photon counting is a highly sensitive technique for measurement of lowlevels of electromagnetic radiation. In photon counting detection,current produced by a photon hitting the anode of a PMT with sufficientenergy to begin an avalanche of electrons is tested by a discriminatorcircuit to distinguish between random electronic noise and true signal.At such light levels, the discreteness of photons dominates measurementand requires technologies that enable distinguishing electrical pulsesthat are photon-induced from dark-current impulses that originate in thedetector (e.g. a PMT) from other causes.

In previous applications of photon counting, the dynamic range ofdetection was restricted by the ability of the detector to discriminatebetween photons closely spaced in time. Additionally, the signal tonoise ratio in photon counting is also a function of the lightintensity. Assume a steady light flux incident on a photocathodeproducing m photoelectrons per second During any one second, the lightincident on the photocathode is, on average, m photoelectrons with astandard deviation of m^(1/2). The signal to noise ratio in suchmeasurements is:

S/N=m/m ^(1/2) =m ^(1/2)  (1)

Depending upon their frequency and energy, individual photoelectrons canbe counted with a detector of sufficient gain, but the precision of anymeasurement can never be better than the limit imposed by equation (1).In its simplest form, a practical photon counting instrument consists ofa fast amplifier and a discriminator set to a low threshold relative tothe input, typically −2 mV, which has been found empirically tocorrespond to the optimum compromise between susceptibility toelectrical pickup and operating the photomultiplier at excessive gain.

Theoretically less sensitive than photon counting and with greatersensitivity to electronic drift, signal averaging uses photocurrent as adirect measure of the incident light signal. The noise associated withthe photocurrent I_(k), taking the system bandwidth (frequency ofresponse), B, into account, is given by the shot noise formula:

S/N=(I _(k)/2eI _(k) B)^(1/2)  (2)

where e is the electronic charge. The forms of equations 1 and 2 aresimilar, since they refer to the same phenomenon and predict essentiallythe same result. In contrast to photon counting in which the signal isinherently digitized and its dynamic range limited by the speed of thetimer counter, in equation 2 the signal is taken as a continuousvariable of the photocurrent and it is possible to obtain a much largerdynamic range. In practice, however, the analog-to-digital conversionprocess severely limits the dynamic range owing to the slow responsetimes associated with AID converters having more than 16-bit resolution.

In general purpose fluorescence detection instruments, the light sourcecan be a quartz halogen lamp, a xenon lamp or similar gas dischargelamp, a photodiode or one of many types of lasers. Typically the sampleis exposed to continuous illumination which maintains a relativelystable percentage of the total number of fluorophores in an excitedstate. In these instruments, the cross-sectional dimension of the samplewhich is illuminated is principally determined by slits. In more complexinstruments, including any using imaged light, confocal optics or pointsource illumination, the exciting light beam is shaped and focused bylenses and mirrors onto a single point and a single focal plane in thesampler

SUMMARY

According to various embodiments of the present invention, a wavelengthand area scanning fluorescence spectrophotometer is provided thatincludes an excitation double monochromator, a coaxialexcitation/emission light transfer module, an emission doublemonochromator, a high speed timer-counter circuit board, and a precisionx-y-z mounting table for use in positioning a sample relative to thefocal plane of the excitation light.

Operations of each are directed and coordinated through a timer-counterboard. Each monochromator may include a pair of holographic concavegratings mounted to select a desired band of wavelengths precisely fromincoming broadband light. Selected excitation light is directed into asample well or onto a two dimensional surface such as a polyacrylamidegel or microscope slide by a light transfer module that includes acoaxial excitation mirror positioned to direct excitation light directlyinto a well of a multi-well plate or onto a particular area of a gel,microscope slide, or microarray. Emitted light that exits theilluminated area or the sample is collected by a relatively largefront-surfaced mirror. The collected emission light is wavelengthselected by the emission double monochromator. Both monochromatorscontain three precision matched apertures that are positioned torestrict unwanted wavelengths while simultaneously creating a “nearpoint” source of the desired wavelengths for the succeeding stage of theoptical path. Emission light that has been isolated in this way isprojected onto the photodetector and analyzer module which converts thereceived energy into a digital representation of the fluorescenceintensity of the sample.

One embodiment includes a fluorescence spectrophotometer system having alight source; a first double monochromator operating to separate andoutput selected wavelengths of light from the light source as excitationlight; a light transfer module for directing substantially all of theexcitation light directly onto a sample and for collecting, focusing,and directing fluorescent or luminescent light from the sample asemission light; a second double monochromator operating to separate andoutput selected wavelengths of the emission light; and a photodetectorand analyzer for detecting the selected wavelengths of emission lightand outputting an indication of such detection.

Another embodiment includes a double monochromator having an entranceaperture for accepting light; a first optical grating positioned tointercept and to disperse at least part of the light accepted throughthe entrance aperture; a first selection aperture positioned tointercept part of the light dispersed by the first optical grating andto select and pass a narrowed range of wavelengths from such dispersedlight; a second optical grating positioned to intercept and disperse atleast part of the light passed through the first selection aperture; anda second selection aperture positioned to intercept part of the lightdispersed by the second optical grating and to select and pass anarrowed range of wavelengths from such dispersed light.

Yet another embodiment includes a light transfer module having anexcitation mirror, positioned substantially coaxial with an area to beilluminated, for directing incoming light to illuminate the area; and anemission mirror, positioned substantially coaxial with the area that hasbeen illuminated and in off-axis alignment with the excitation mirror,for collecting, focusing, and directing light emitted by the area uponillumination.

Another embodiment includes a photon counting photodetector and highspeed timer-counter board which largely eliminates instrument drift,provides great sensitivity while enabling high frequency discriminationof photons for maximum resolution and quantitation and, concurrently,provides significantly greater dynamic range.

In accordance with another embodiment, a fluorescence spectrophotometersystem additionally includes an optical polarizing filter operative torestrict the excitation light to plane polarized excitation light and anoptical filter holder selectively operative to insert the opticalpolarizing filter into the path of the excitation light. The opticalfilter and the optical filter holder may be incorporated into the firstdouble monochromator described above; alternatively, the optical filterand the optical filter holder may be incorporated into the lighttransfer module.

Additionally, the foregoing system may also include a first polarizingfilter operative to transmit emission light in a plane which is parallelto the plane of the polarized excitation light, a second polarizingfilter operative to transmit emission light in any plane which is notparallel to the plane of the polarized excitation light, and apolarizing filter holder selectively operative to insert one of thefirst polarizing filter or the second polarizing filter into the path ofthe emission light. As with the optical filter, the first polarizingfilter, the second polarizing filter, and the polarizing filter holdermay alternatively be incorporated into the second double monochromatoror the light transfer module. When incorporated into the light transfermodule, such emission polarizer filters may be interposed between theilluminated area and the emission mirror; i.e. upstream of the emissionmirror.

In other embodiments set forth in more detail below, a fluorescencespectrophotometer system may incorporate a light source comprising aspherical concave reflector system having interchangeable apertures, thereflector system being telecentric at both ends and fully corrected forthird order aberrations, a first multiple-grating monochromator havingan entrance aperture, the first multiple-grating monochromator beingoperative to separate light imaged onto the entrance aperture from thelight source into a plurality of wavelengths and to output selectedwavelengths as excitation light, and a light transfer module comprisinga first reflection surface operative to direct substantially all of theexcitation light directly onto a sample and a second reflection surface,the second reflection surface being a compound parabolic reflectivesurface and operative to collect, focus, and direct light emitted fromthe sample as fluorescent or luminescent light.

In still other embodiments, elements of the system including the lighttransfer module, the second multiple-grating monochromator, and thephotodetector and analyzer are operative to analyze more than onefluorescent compound in the sample. Additionally or alternatively, aspectrophotometer system may comprise means for translating a sample orsample holder, such as a microwell plate, for example, relative to thelight transfer module allowing analysis of samples from selected ones ofa plurality of wells in the microwell plate.

In accordance with another aspect as set forth in detail below, a methodof analyzing a sample generally comprises providing excitation lightfrom a light source, directing the excitation light through a firstdouble monochromator, transmitting the excitation light to the samplethrough a light transfer module, employing the light transfer module toobtain light emitted by the sample, directing the light emitted by thesample to a second double monochromator, and analyzing light output bythe second double monochromator. The method may be employed to detectand to analyze more than one fluorescent compound in a single sample.

The foregoing and other aspects of various embodiments of the presentinvention will be apparent through examination of the following detaileddescription thereof in conjunction with the accompanying drawings.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of an embodiment of a fluorescencespectrophotometer system

FIG. 2A is a schematic diagram of an embodiment of a doublemonochromator.

FIG. 2B is a schematic diagram showing one embodiment of a tension bandactuator mechanism for pivoting the gratings of the double monochromatorshown in FIG. 2A.

FIG. 3 is a simplified schematic diagram of one embodiment of the lighttransfer module shown in FIG. 1.

FIG. 4 is a schematic diagram of a simplified version of a fluorescencespectrophotometer constructed and operative in accordance with thepresent invention.

FIG. 5A shows a first alternative dual path embodiment of a fluorescencespectrophotometer system.

FIG. 5B shows a second alternative dual path embodiment of afluorescence spectrophotometer system.

FIG. 6 is a schematic diagram of an embodiment of a fluorescencespectrophotometer system including fluorescence polarization.

FIG. 7 is a simplified diagrammatic view of one embodiment of apolarization analyzer constructed and operative in accordance with oneembodiment of the present invention.

FIG. 8 is a simplified cross-sectional diagram of an illumination system

FIG. 9 is a simplified cross-sectional diagram of one embodiment of alight transfer module incorporating a compound parabolic concentrator.

FIG. 10A is a simplified cross-sectional diagram of the interior of alight transfer module incorporating a compound parabolic concentrator.

FIG. 10B is a simplified cross-sectional diagram of one embodiment of alight transfer module incorporating emission polarization upstream of anemission mirror.

FIG. 11 is a simplified flow diagram illustrating one embodiment of amethod of analyzing a sample.

Unless otherwise noted, like reference numerals and designations in thevarious drawings indicate like components.

DETAILED DESCRIPTION

FIG. 1 is a schematic diagram of one exemplary embodiment of afluorescence spectrophotometer system constructed and operative inaccordance with the present invention. A broadband light source 100illuminates a mirror 102, which is suitably curved to focus the lightonto a double monochromator 104. In alternative configurations, a narrowband light source such as a photodiode or a laser may be substituted forthe broadband light source depicted in FIG. 1; those of skill in the artwill appreciate that such a narrow band light source may be implementedin conjunction with the any or all of the fluorescence spectrophotometersystems and monochromator configurations set forth in detail below.

In some alternative embodiments, for example, light source 100 maycomprise a halogen cycle tungsten filament lamp or other suitable lampoperative to transmit light to a spherical concave reflector systemhaving interchangeable apertures; as described below, such a reflectorsystem may be telecentric at both ends of the optical train and fullycorrected for third order aberrations.

Light of the desired wavelength is passed by an excitation doublemonochromator 104 to a light transfer module (LTM) 106. The LTM 106directs the excitation light from monochromator 104 onto a sample 108,which can be, for example, one well of a microwell plate or one lane ofa 1-D polyacrylamide gel. Any resulting fluorescent or luminescent lightemitted by the sample may be collected by LTM 106 as set forth below,LTM 106 may direct the light to the entrance aperture of an emissiondouble monochromator 110. In operation, monochromator 110 may beselectively adjusted to pass wavelengths from the emission spectrum ofthe fluorophore(s) in the sample, and is operative to direct thosewavelengths to a photodetector 112, which measures the energy of theemitted light.

Photodetector 112 may be any suitable photosensitive device, includingbut not limited to a photomultiplier tube (PMT), a phototransistor, or aphotodiode. The electronic output of photodetector 112 may be applied toan automatic processing unit 114, which generates a signal (which may bestored in a numerical form suitable for further analysis) indicatingdetection of the selected emission. Automatic processing unit 114 maybe, for example, a personal computer having a data collection interfaceto the spectrophotometer system. In general, all of the elements of theoptical pathways in the instrument depicted in FIG. 1, including doublemonochromators 104 and 110 and the LTM 106, may be isolated inlight-tight boxes coated internally with non-fluorescent absorptivematerial to minimize reflectance and light from other sources such asroom light.

FIG. 2A is a schematic diagram of an embodiment of a doublemonochromator configured and operative for use in embodiments of afluorescence spectrophotometer system The illustrated configuration maybe used for either the excitation double monochromator 104 or theemission double monochromator 110 illustrated in FIG. 1.

Broadband light is introduced through an entrance aperture 200 in thedouble monochromator and reflects off the front surface of a firstholographic concave grating 202 that is pivotable about an axis 203. Useof front surface reflection enhances the efficiency of a doublemonochromator by avoiding absorption of light within an optical supportstructure, such as in a rear surface reflection glass mirror. Use of aconcave grating allows incident light to be dispersed into selectablewavelengths without the use of supplemental collimating mirrors. Thisdesign makes it possible to eliminate two collimating mirrors pergrating used in conventional dual monochromator instruments. Use of aholographic grating also reduces astigmatic aberrations, and thusdecreases the amount of light of unwanted wavelengths (“stray light”).

Each double monochromator has three apertures: an entrance aperture 200,through which light first enters the monochromator; an internal (or“first”) selection aperture 206; and an exit (or “second”) selectionaperture 216, through which light exits the monochromator. The firstconcave grating 202 reflects wavelengths of the incoming light as afirst spatially dispersed beam 204. Each wavelength of this firstspatially dispersed beam 204 is reflected at a unique angle relative toother wavelengths. Pivoting first concave grating 202 about its axis 203enables a selected band or range of wavelengths 208 to be directedthrough the internal selection aperture 206 within the monochromatorhousing.

The selected range of wavelengths 208 is then reflected off a secondholographic concave grating 210 that is pivotable about an axis 211.Second concave grating 210 reflects the selected range of wavelengths208 of the first spatially dispersed beam 204 as a second spatiallydispersed beam 212. Pivoting second concave grating 210 about its axis211 enables a selected narrow band of wavelengths 214 to be directedthrough exit selection aperture 216.

The dimensions of selection apertures 206 and 216 determine the selectedwavelengths of light leaving the monochromator. Wider apertures allowmore light energy to pass through a monochromator, but the lightincludes a broad range of wavelengths; narrower apertures reduce theamount of light passing through a monochromator but narrow the selectedrange of wavelengths. The use of wider apertures increases thesensitivity of detection, which may be beneficial in measurements oftotal fluorescence in an area or volume (e.g. measurements made inmicrowells). In contrast the use of narrow apertures increases thespatial resolution of a monochromator, which may be beneficial indiscriminating between different fluorophores at particular locations(e.g. measurements of bands separated in a polyacrylamide gel). That is,the smaller the aperture dimensions, the smaller the area of detectionat the sample. In the case of laser light sources and pinhole apertures,for example, the area of excitation at any given moment is a point thatcorresponds to a particular data pair representing fluorescent intensityand position.

It is generally known by those of skill in the art of optical systemsthat each optical component reduces the efficiency of light throughput.Advantages of the configuration shown in FIG. 2A include, but are notlimited to, elimination of collimating mirrors for redirecting lightwithin the double monochromator, such as is the case with traditionalmonochromators. In the exemplary embodiment, only two light directingelements (the first and second holographic concave gratings 202, 210)are required, thus improving overall light efficiency.

In the configuration illustrated in FIG. 2A, the first concave grating202 and the second concave grating 210 may be pivoted oppositely and intandem by a suitable mechanism A mechanism according to one embodiment(illustrated in FIG. 2B) implements a tension band actuator mechanismfor pivoting gratings 202, 210. In the FIG. 2B configuration, gratings202, 210 are mounted coaxially with pivot wheels 250, 252, respectively.One end of a lever arm 254 is connected to pivot wheel 250; the otherend of lever arm 254 is coupled to a screw drive mechanism 256. Leverarm 254 traverses along threaded rod 258 as the rod 258 is rotated by amotor 260.

Lever arm 254 movement along rod 258 causes pivot wheel 250 to rotate.Pivot wheel 250 is connected to pivot wheel 252 by a tension band 262.In accordance with the FIG. 2B embodiment, tension band 262 may be a0.003 inch stainless steel band Tension band 262 causes pivot wheel 252and grating 210 to counter-rotate synchronously and in tandem with pivotwheel 250 and grating 202. A spring 264 connected between a housing (notshown) and pivot wheel 252 maintains tension in tension band 262. Theposition (i.e. angular orientation) of gratings 202,210 may bedetermined and controlled by a microcontroller connected to pivot wheelsensor 266 and screw drive sensor 268.

The FIG. 2B embodiment is provided by way of example only, and not byway of limitation; it will be appreciated that alternative mechanismsexist for pivoting gratings 202,210 either synchronously or otherwise.For example, each grating 202,210 may be provided with a respectivepivot mechanism such that the angular orientation of gratings 202,210relative to each other may be independently selectable. Additionally,alternative means for coupling gratings 202,210 for synchronous andcomplementary rotation are within the scope and contemplation of thepresent disclosure.

FIG. 3 is a schematic diagram of one embodiment of the LTM 106 shown inFIG. 1. Excitation light enters LTM 106 through an entrance aperturewhereupon it is directed, either with or without one or more mirrors300, to a coaxial excitation mirror 302. The coaxial excitation mirror302 may be flat, or include appropriate curvature to focus or todisperse light as desired Coaxial excitation mirror 302 is positioned todirect the excitation light directly onto the sample 108. Moreparticularly, coaxial excitation mirror 302 may be positioned somewhatoff-axis with respect to sample 108, but should be positioned so thatsubstantially all of the excitation light strikes the sample to achievemaximum illumination.

In one application, each well of a multi-well plate can be positionedbeneath the coaxial excitation mirror 302 by X-Y translation of eitherLTM 106, the multi-well plate, or both. In another application involvingmonochromators equipped with optional microscope optics, differentregions of intact biological cells that have been mounted on glassslides or culture plates can be separately imaged using optical elementsin the light path and by positioning the sample beneath coaxialexcitation mirror 302 by X-Y-Z translation of either LTM 106, the glassslides or culture plates, or both Fluorescence emission from one or morefluorophores in a sample may be collected by a coaxial emission mirror304. Coaxial emission mirror 304 may be concave so as to focus anddirect the emission light, either directly or by one or more lightdirecting mirrors 306, out of an exit port or aperture of LTM 106. Inthe embodiment shown in FIG. 3, emission light exits LTM 106 from thesame side that excitation light enters LTM 106. However, differentplacements of the entrance and exit apertures may be used by suitableplacement of light directing mirrors 300, 306.

In this context, “coaxial” generally refers to the position andorientation of mirrors 302,304 relative to an area to be illuminated (inthe case of excitation mirror 302) and relative an area that is emittingfluorescent or luminescent light (in the case of emission mirror 304).The coaxial placement of excitation mirror 302 with an area to beilluminated, as well as the coaxial placement of emission mirror 304with an area emitting light, combine to ensure that a high percentage ofexcitation light is directed onto the sample within a well 108, forexample, and that a high percentage of the fluorescent or luminescentlight emitted from the opening of well 108 is collected for analysis. Insome embodiments, emission mirror 304 may be positioned slightlyoff-axis relative to excitation mirror 302 to avoid interference in theoptical train.

In a particularly efficient embodiment, all of the mirrors within LTM106 comprise front, or “first,” surface mirrors. First surface mirrorshave a reflective material, such as aluminum or other reflective metal,for example, coated onto the surface of a substrate, such as glass orceramic; incident light is directed onto the reflective coating. Sincethe coating serves as the reflective surface, incident light does notpenetrate the substrate as in an ordinary second surface mirror.Accordingly, such first surface mirrors are substantially more efficientthan traditional second surface mirrors.

It will be understood by those of ordinary skill in the art that anumber of different reflecting mirrors may be used to direct lightwithin LTM 106, as needed. As noted above, however, it is generallydesirable to minimize the number of such reflecting surfaces in order toimprove efficiency of LTM 106. In the FIG. 3 embodiment, excitationmirror 302 may be an elliptical mirror approximately 6×9 millimeters indimension, while the emission mirror 304 may be approximately 75millimeters in diameter. Other dimensions can be used and generally willvary with the dimensions of the overall instrument.

FIG. 4 is a schematic diagram of a simplified embodiment of afluorescence spectrophotometer. As in the embodiment described abovewith reference to FIG. 1, excitation wavelengths may be selected from abroadband light source 100 by means of an excitation doublemonochromator 104. The excitation light is directed by a coaxialexcitation mirror 302 to a sample within a well 108. Fluorescent andluminescent emission light may be collected and focused by a coaxialemission mirror 304 that is in reflective alignment with the coaxialexcitation mirror 302. Emission mirror 304 directs the collected andfocused light into an emission double monochromator 110. As describedabove, monochromator 110 directs one or more selected wavelengths ofemission light to a photodetector and analyzer module 112 for countingand analysis. This embodiment minimizes the number of reflectivesurfaces within LTM 106.

The compact configuration of the embodiment shown in FIG. 4 allows foruse of the instrument as a dual path spectrophotometer. That is, asample can be illuminated with excitation light either from the openside of well 108 or from the bottom side of well 108 as set forth below.

FIG. 5A shows a first alternative dual path embodiment of a fluorescencespectrophotometer system. One or both of the light source 100 and theexcitation double monochromator 104 optionally may be translated from afirst position (illustrated in FIG. 5A by dashed lines) to a positionbelow a multi-well plate such that excitation light impinges upon abottom-illumination coaxial excitation mirror 302′, which directsexcitation light through the transparent bottom substrate of a well 108.Alternatively, if light source 100 is not translated, light directingmirrors may be interposed to direct light through excitation doublemonochromator 104. Fluorescence emissions emanating from the opening ofwell 108 may be collected by emission mirror 304. When the system isconfigured for bottom-illumination, the top-illumination excitationmirror 302 (illustrated in FIG. 4, but not shown in FIG. 5A) may beremoved from the light path in order to maximize the amount offluorescent and luminescent light collected by the emission mirror 304.Alternatively, top-illumination excitation mirror 302 can be left inplace. In either case, bottom-illumination excitation mirror 302′ is indirect alignment (as opposed to reflective alignment) with emissionsmirror 304.

FIG. 5B shows a second alternative dual path embodiment of afluorescence spectrophotometer system. In this configuration implementedfor bottom illumination, a set of one or more redirection mirrors 303A,303B may be interposed into the light path from excitation doublemonochromator 104 in order to intercept and to direct excitation light.The intercepted excitation light is redirected to impinge upon abottom-illumination coaxial excitation mirror 302′, which directs theexcitation light through the transparent bottom substrate of well 108.The top-illumination coaxial excitation mirror 302 may be left in place,as shown, or moved out of position when the redirection mirrors 303A,303B are moved into position. Such movement may be accomplished, forexample, by translation (e.g. along an axis into or out of the page) ofa carriage on which all four mirrors 302, 303A, 303B, 302′, are mounted.As with the FIG. 5A embodiment, bottom-illumination excitation mirror302′ is in direct alignment (as opposed to reflective alignment) withemission mirror 304.

In all of the configurations shown in FIGS. 4, 5A, and 5B, additionalredirection mirrors may be used as required to guide light to selectedlocations within the instrument.

According to an embodiment, a timer-counter board that operates at afrequency in excess of 100 MHz may be used. The photodetector andanalyzer module (designated by reference numeral 112 in FIGS. 4, 5A, and5B) may be configured to respond to photons at frequencies as high as 30MHz. The dynamic range of this system ranges from 0 to more than 30×10⁶photons, thereby eliminating the dynamic range limitations whichpreviously restricted the use of photon counting in fluorescencedetection. If the observed current from the PMT is greater than aprescribed threshold established at the photodetector, a 5 voltelectrical pulse having a duration of approximately 2 nanoseconds isproduced; the timer-counter circuit enumerates these pulses as afunction of time that is, establishes a direct quantitation for photonsper unit time.

Using a spectrophotometer according to one embodiment, a pre-castpolyacrylamide gel containing fluorescently labeled nucleic acids orproteins was placed on a flat plate in the positioning mechanismdirectly under the LTM. With the excitation and emission monochromatorsset at wavelengths suitable for the fluorescent labels, the gel wastranslated (in the X and Y directions) under the LTM until all of thegel area had been traversed. At each point of this travel a fluorescentreading was made and stored as a two dimensional array representing thefluorescent emission at each point of the gel. The distance between thepoints was adjusted to yield the best response for a given dataacquisition time. In the actual experiments, the gel was also scanned as“lanes” representing the path of electrophoresis from the sample well atthe top of the gel to the base of the gel because the fluorophores in anelectrophoresis gel are arrayed in a line rather than as a point. Eachsuch lane was scanned from the sample well to the bottom of the gel toachieve a substantial reduction in overall data collection time. As areference for background, a blank lane was scanned and the datasubtracted on a point-by-point basis from the corresponding data forlanes containing fluorophores. The corrected data were then analyzed intwo ways. In one analysis, an image was constructed of the original gelwhich was compared to standard laboratory photographs of the same gelfor evaluation of standard gel parameters such as migration distance,separation of molecules, and concentration of molecular species asdetermined separately by the digital image and the film. In the otheranalysis, a “densitometry” plot equivalent to those made for gel lanesfrom autoradiography films using flat bed scanning detectors wascreated. From the database relating fluorescence intensity of a lane,the center of each fluorescent band was identified and a cross sectionalgraph of fluorescent intensities as a function of migration distance wasprepared. From the use of gels prepared with different but known amountsof the same fluorescent labeled nucleic acids, a standard curveestablishing lower and upper limits of sensitivity, resolution, andoverall dynamic range for gel detection were determined.

The “square intensity point spread function” and the “long penetrationdepth” properties of one and two photon absorption processes have beenrecognized as important features in future developments in fluorescencedetection. Both are accomplished by focusing a femtosecond short pulselaser onto a focal plane in a sample to be studied for fluorescence. Ina spectrophotometer according to another embodiment, a laser beam froman appropriate laser was substituted for the quartz halogen or xenonlight source used for excitation. The laser beam was used to illuminatethe entrance aperture of the excitation monochromator. Additionalmodifications where needed in some cases including, for example, the useof one or two pinhole apertures rather than the standard rectangularslits, and the insertion of an objective lens to focus the light afterit had passed through the monochromator. The fluorescence emission wascollected through the light transfer module as previously and thefluorescence measured as a function of time, or in the cases of imageformation and area scanning, as a function of time and position of thelight transfer module over the sample as described in the gel analysisabove. In this configuration, a pinhole exit aperture on the excitationmonochromator was used to image light onto a sample, and each point ofthe image used to excite fluorescence. Moving the sample position in anx-y-z fashion enabled scanning of areas of the sample to create an imageor database. For confocal microscopy, two pinhole apertures wererequired as described below. For multi-photon applications, only asingle pinhole aperture was used as the exit aperture of the excitationmonochromator. For two-photon excitation, a microlens array could beused if needed to focus the beam for high transmittance. In general theconfiguration was epi-fluorescent although in certain polarizationapplications, excitation was from the bottom and emission light wascollected from the top.

In yet another embodiment, a fluorescence spectrophotometer systemconstructed and operative in accordance with the present invention wasapplied in the creation of a scanning fluorescence polarizationdetector. Fluorescent molecules in solution, when excited with planepolarized light, will emit light back into a fixed plane (i.e. the lightremains polarized) if the molecules remain stationary during thefluorophore's period of excitation (excited state lifetime). Moleculesin solution, however, tumble and rotate randomly, and if the rotationoccurs during the excited state lifetime and before emission occurs, theplanes into which light is emitted can be very different from the planeof the light used for the original excitation.

The polarization value of a molecule is proportional to its rotationalrelaxation time, which by convention is defined as the time required fora molecule to rotate through an angle of 68.5°. Rotational relaxationtime is related to solution viscosity (Ti), absolute temperature (T),molecular volume (V), and the gas constant (R):

Polarization value∂Rotational Relaxation Time=3ρV/RT

If viscosity and temperature are constant, the polarization is directlyrelated to molecular volume (molecular size), which, in generalcorrelates well with molecular weight. Changes in molecular volumeresult from several causes, including degradation, denaturation,conformational changes, or the binding or dissociation of two molecules.Any of these changes can be detected as a function of changes in thepolarization value of a solution. Specifically, a small fluorescentmolecule which rotates freely in solution during its excited statelifetime can emit light in very different planes from that of theincident light. If that same small fluorophore binds to a largermolecule, the rotational velocity of the small molecule decreases andthe effect is detected as a decrease in the polarization value.Measurement of the effect requires excitation by polarized light whichcan be obtained using a laser or through light selection using apolarizing filter which only transmits light traveling in a singleplane. In one experiment, light of a defined wavelength obtained fromone embodiment of an excitation monochromator operative in accordancewith the present invention was further refined by passing the lightthrough a polarizing filter (designated the “polarizer”) to obtainmonochromatic, plane polarized light for excitation (for the presentpurposes designated “vertically polarized light”). Concurrently, thelight path for collecting the emission light was similarly modified byselective introduction of one of a pair of polarizing filters(designated the “analyzers”), one of which can be rotated to a positionvertical to the plane of the excitation light, whereas the other ofwhich can be rotated to a position horizontal to the plane of theexcitation light. When a fluorescent sample in solution was introducedinto the light path between the polarizer and the analyzers, only thosemolecules which were oriented properly to the vertically polarized planeabsorbed light, became excited, and subsequently emitted light. Byselecting the appropriate analyzing filter to be inserted into theemission light path, the amount of emitted light in the vertical andhorizontal planes can be measured and used to assess the extent ofrotation of the small fluorescent molecule in the solution before andafter binding to a larger molecule. FIG. 6, described below, illustratesa system which may facilitate the forgoing experiment.

In yet another application, a fluorescence spectrophotometer system wasutilized in confocal microscopy—a method for eliminating one of thefundamental difficulties of fluorescence microscopy, namely, thereduction in spatial resolution at the focal plane of the microscopeowing to out-of-focus light. A spectrophotometer according to anotherembodiment was used to create a confocal microscope from the embodimentessentially as described under laser excitation above to focus a lightimage on whole cell mounts and to achieve both multiple and singlephoton excitation of the fluorescent labels in a sample. Fluorophores inplanes out of the focus were not illuminated and did not fluoresce.

In confocal imaging, apertures were used in both the excitation andemission light paths in order to focus a cone of light through thespecimen and in the emission light path in order to eliminate scatteredand out-of-plane fluorescence. The development of mode locked dye lasershas made simultaneous multi-photon excitation practical because suchlasers are capable of delivering the available excitation energy to afocal spot in very brief pulses and with sufficient energy to achievetwo photon excitation. In multi-photon imaging, the focal spot providedby the laser excites a sufficiently small volume that, when used inconjunction with the light transmission module described above, makes itpossible to collect all emission light without a second pinhole apertureon the emission side. No emission aperture changes were necessary.

FIG. 6 is a schematic diagram of an embodiment of a fluorescencespectrophotometer system including fluorescence polarization as notedbriefly above. In the FIG. 6 embodiment, light exiting the excitationmonochromator 104 may be plane polarized. In that regard, an excitationpolarizing filter 602 may be operative to polarize the excitation lightin a selected orientation. The polarized excitation light is transferredto the sample well 108 via LTM 106. Resulting fluorescent andluminescent light emitted by the sample is collected by LTM 106, whichdirects emission light to a polarization analyzer 604. In operation,polarization analyzer 604 may determine if the light emitted from well108 has been rotated from its original orientation 602. Such rotationmay provide information about the sample in well 108, for example, thespin and/or size of molecules in the sample. Analyzer 604 transmits theemission light to the entrance aperture of emission double monochromator110 as described above with reference to the embodiment shown in FIG. 1.

Excitation polarizing filter 602 may generally be implemented as anoptical filter operative to restrict the excitation light to planepolarized excitation light oriented in a selected plane, as is generallyknown in the art. An optical filter holder (not shown) may be employedselectively to insert the optical polarizing filter 602 into the path ofthe excitation light.

FIG. 7 is a simplified diagrammatic view of one embodiment of apolarization analyzer 700 constructed and operative in accordance withone embodiment of the present invention. Analyzer 700 generallycorresponds, to and incorporates all of the functionality of, analyzer604 described above with reference to FIG. 6. Analyzer 700 may includean aperture 702 operative to admit emission light (from LTM 106 asindicated in FIG. 6) and two emission polarizing filters 704 and 706,which may be disposed on a filter holder such as plate 708, for example.A first emission polarizing filter 704 has a polarization parallel tothat of the excitation polarization filter 602, and hence passes lighthaving the original orientation produced by the excitation polarizingfilter 602. A second emission polarizing filter 706, on the other hand,has a polarization perpendicular to that of excitation polarizing filter602, and hence blocks light having the original orientation A motorizedslider 710 or other suitable mechanism may slide filter plate 708between two positions: a first position in which the first (parallel)polarization filter 704 is aligned with aperture 702 in the light train,and a second position in which the second (perpendicular) polarizationfilter 706 is aligned with aperture 702 in the light train.

Two measurements may be taken for each sample. For the firstmeasurement, the motorized slider 710 slides the filter plate 708 intothe first position and any emission fluorescence from the sample may bepassed through the parallel filter 704. For the second measurement, themotorized slider 710 slides the filter plate into the second position,and any emission fluorescence from the sample may be passed through theperpendicular filter 706. The amount of rotation, if any, may bedetermined by comparing the two measurements. For example, if thepolarization of the excitation light has not been rotated in the sample,no emission fluorescence should be detected in the second measurement,since all of the light in the original orientation would be blocked bythe perpendicular filter 706.

Though polarization analyzer 700 has been illustrated and described as adiscrete component (e.g. reference numeral 604 in FIG. 6) of afluorescence spectrophotometer system, it will be appreciated that thecomponents detailed in FIG. 7 or their equivalents, as well as thepolarization analyzer functionality, may alternatively be incorporatedinto the emission side of LTM 106 or into monochromator 110 illustratedin FIG. 6. As set forth in detail below, some embodiments incorporateemission polarization analyzer functionality in the LTM upstream of theemission mirror in the light train. Similarly, an excitation polarizingfilter (reference numeral 602 in FIG. 6) may be incorporated intomonochromator 104 or the excitation side of LTM 106.

FIG. 8 is a simplified cross-sectional diagram of an illumination systemIllumination system 800 may generally correspond to light source 100illustrated in FIGS. 4, 5A, and 5B. Illumination system 800 may beoperative to image the filament of a light source or lamp 801 onto theentrance aperture 200 of an excitation monochromator such as representedby reference numeral 104 in FIG. 1. In the FIG. 8 embodiment, a firstspherical reflection surface 802 and a second spherical reflectionsurface 803 cooperate to form a spherical concave reflector system, inthis case an Offner 1:1 afocal relay. Spherical surfaces 802,803 may beused to correct for all third order aberrations; the spherical concavereflector system is telecentric at both ends of the optical train.

As is generally known in the art, lamp 801 may include a rear mirror 804operative to reflect the flux from the rear of the filament forwardthrough the system Additionally or alternatively, an aperture wheelhaving interchangeable apertures (not shown) may be inserted into thelight path Rotation of the aperture wheel enables selection of one of aplurality of apertures to be inserted into the light path illustrated inFIG. 8, for example; accordingly, the cone angles of the flux incidenton entrance aperture 200 may be selectively adjusted in accordance withsystem requirements.

In that regard, means for selectively inserting one of a plurality ofapertures into the path of light moving through the spherical concavereflector system of illumination source 800 may be included Mechanismssuch as linear actuators, gears for rotating an aperture wheel and thelike are contemplates Various methods of interposing one or moreapertures into an optical train are known in the art.

FIG. 9 is a simplified cross-sectional diagram of one embodiment of alight transfer module incorporating a compound parabolic concentrator.As indicated by the arrow in FIG. 9, excitation light may be admitted toLTM 106 (generally represented by the dashed lines in FIG. 9) from theleft side of the diagram, and is directed through an aperture 902 byexcitation mirror 302 (see FIGS. 4, 5A, and 5B), which is appropriatelymounted as shown, to illuminate a sample. A compound parabolicconcentrator (CPC) 901 may receive emission light (i.e. fluorescent orluminescent light emitted from the illuminated area containing sample)through aperture 902.

In accordance with this embodiment, CPC 901 may comprise a polished,reflective surface operative to collect the flux radiated from theilluminated sample and to concentrate emission light for reflection byan emission mirror (not shown in FIG. 9) such as described in detailabove.

As noted above, the various components of the emission polarizationanalyzer (see FIGS. 6 and 7) may be interposed between CPC 901 and theemission mirror, incorporating the functionality of a polarizationanalyzer into LTM 106. This arrangement may substantially reduce theinfluence of the LTM 106 itself on the polarization state of theemission light.

FIG. 10A is a simplified cross-sectional diagram of the interior of alight transfer module incorporating a compound parabolic concentrator,and FIG. 10B is a simplified cross-sectional diagram of one embodimentof a light transfer module incorporating emission polarization upstreamof an emission mirror. The views depicted in FIGS. 10A and 10B areparallel to the light train and are taken at lines 10A and 10B,respectively, shown in FIG. 9; i.e. emission light directed throughaperture 902 of CPC 901 in FIG. 10A passes through the polarizer unit910 in FIG. 10B before being directed by an emission mirror to anemission double monochromator as described above.

In the FIG. 10A embodiment, an aperture 903 admits excitation light froman excitation double monochromator into LTM 106 as described above withreference to FIG. 9. As noted above, emission light emitted by theilluminated sample passes through aperture 902 and is directed by thepolished interior surface of CPC 901 to an emission mirror.

Polarizer unit 910 illustrated in FIG. 10B may be interposed between CPC901 and an emission mirror, such that emission light may be polarized orotherwise filtered before it impinges on the emission mirror. Inoperation, an actuator or drive motor 907, for example, coupled to afilter holder or wheel 904 may enable selection of one of a plurality ofpolarizing filters (represented by reference numerals 905A and 905B) orother filters (represented by reference numeral 906). One or more slots(represented by reference numeral 908) in wheel 904 may be provided withno filter at all. Accordingly, a filter holder such as wheel 904 may beselectively operative to insert one of a plurality of polarizing orother filters into the path of the emission light. The quantity, nature,and polarization plane of the various filters incorporated in wheel 904may be selected as a function of overall system requirements.

It will be appreciated that alternative or additional mechanisms may beimplemented to enable selection of filters in polarizer unit 910. Lineartranslation of a filter plate such as described above with reference toFIG. 7, for example, may be appropriate and equally effective, dependingupon size and operational requirements of the LTM in the context of theoverall spectrophotometer system.

It will also be appreciated that the foregoing descriptions of thedrawing figures are exemplary only, and that the disclosed embodimentsare susceptible to modifications and alterations which may improveoverall system efficiency. For example, rotating aperture wheels andappropriate mountings may additionally be implemented in the LTM 106 ofFIG. 9; apertures may be selected in accordance with the dimensions of asample well, for instance, to adjust excitation light passed throughaperture 902 such that stray light may be minimized.

In one embodiment of a double monochromator such as depicted in FIG. 2A,for example, baffles may be incorporated to reduce stray lightoriginating from diffracted flux which does not pass through the firstselection aperture 206. Spectroradiometer measurements of monochromatordesigns without baffles have revealed that considerable flux which ishalf the desired wavelength may be transmitted through the firstselection aperture; for example, if a conventional excitationmonochromator is set to pass light having a wavelength of 600 nm, thenflux having a wavelength of 300 nm may also be transmitted through theselection aperture as second order aberrations. Accordingly, reductionof stray light through implementation of baffles may represent animportant determinant with respect to the purity of the light output bythe monochromator.

FIG. 11 is a simplified flow diagram illustrating one embodiment of amethod of analyzing a sample. As set forth in detail above withreference to the system embodiments, a method of analyzing a samplegenerally comprises providing excitation light from a light source(block 1101), directing the excitation light through a first doublemonochromator (block 1102), transmitting the excitation light to thesample through a light transfer module (block 1103), employing the lighttransfer module to obtain light emitted by the sample (block 1104),directing the light emitted by the sample to a second doublemonochromator (block 1105), and analyzing light output by the seconddouble monochromator (block 1106). As noted above, the FIG. 11embodiment may be employed to detect and to analyze more than onefluorescent compound in a single sample.

Several features and aspects of the present invention have beenillustrated and described in detail with reference to particularembodiments by way of example only, and not by way of limitation. Thoseof skill in the art will appreciate that alternative implementations andvarious modifications to the disclosed embodiments are within the scopeand contemplation of the invention. For example, the disclosed doublemonochromators may be implemented in other types of instruments, and theLTM may be employed in filter-based spectrophotometers or other opticalinstruments. As a further example, the LTM may be used to direct inputlight to an area to be illuminated, and efficiently to collect, focus,and direct light emitted (e.g. either by reflection or by fluorescence)from the illuminated area. Accordingly, it is intended that theinvention be considered as limited only by the scope of the appendedclaims.

1. A fluorescence spectrophotometer system comprising: a light source; a first double monochromator comprising two or more gratings and operative to separate light from the light source into a plurality of wavelengths and to output selected wavelengths as excitation light; a light transfer module comprising a first reflection surface operative to direct substantially all of the excitation light directly onto a sample, and a second refection surface operative to direct light that is emitted from the sample as fluorescent or luminescent light; a second double monochromator comprising two or more gratings and operative to separate the fluorescent or luminescent light directed by the light transfer module into a plurality of wavelengths and to output selected wavelengths of the fluorescent or luminescent light as emission light; and a photodetector and analyzer, operative to receive the emission light output by the second double monochromator, to detect the selected wavelengths of the emission light, and to output an indication of the selected wavelengths. 